Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO

Abstract

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.Glucose catabolism in Pseudomonas aeruginosa proceeds by either an oxidative or a phosphorylative pathway ( Fig. 1; reviewed in reference 24). In the direct phosphorylative pathway, glucose is transported into the cell by a periplasmic glucose-binding protein (GLTB)-dependent active transport system. Intracellular glucose is phosphorylated by glucokinase (GLK) and converted to 6-phosphogluconate (6PG) by glucose-6-phosphate dehydrogenase (ZWF). The 6PG from this pathway and from the oxidative pathway ( Fig. 1) is further metabolized to glyceraldehyde-3-phosphate and pyruvate by the Entner-Doudoroff enzymes 6PG dehydratase (EDD) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EDA). GLK, ZWF, EDD, and EDA are strictly co-inducible, and 6PG is thought to serve as the physiological inducer (2,8,19). The glucose transport functions are separately regulated (20).All of the genes known to be required for direct phosphorylative catabolism of glucose to pyruvate and glyceraldehyde-3-phosphate are clustered in the 39-min region of the Pseudomonas aeruginosa chromosome (7,8,36 MATERIALS AND METHODS Bacterial strains and plasmids. All bacterial strains used in this study were derived from prototrophic P. aeruginosa PAO (16) and have been described previously. The edd lesions in strains PFB57 (edd-8 hexR1) and PFB2 (edd4 hexR2) have been described previously (2,8). Both of these mutant strains also were noninducible for the other glucose catabolic enzymes ZWF, GLK, and EDA (2, 8). We have designated these regulatory mutations hexRi and hexR2.Other strains used in this study were PFB9 (edd-1) and PFB52 (edd-2) (2), PFB362 (gltBi) (7), PRP444 (glk-1) (8), PFB98 (zwf-1) (36), PFB103 (zwf-2) (37), and PAO1838 (eda-9001 met-9020) (29).Plasmids in this study w...

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1

“…2) containing a 247-bp XhoI-XmaIII chromosomal fragment was constructed and tested for the hexC effect (56). Activities of GLK, ZWF, EDD, and EDA in crude extracts of P. aeruginosa PAO1(pPZ196) grown in BSM-20 mM lactate were two-to ninefold higher than those of the same enzymes in bacteria without pPZ196 (Table 1), demon- (46,57); and amidase, an enzyme involved in the catabolism of aliphatic amides (19,20). None of these enzyme activities was affected by the presence of pPZ196 in strain PAO1 (Table 1 and data not shown), demonstrating that the hexC effect may be restricted in scope to similarly regulated carbohydrate catabolism enzymes.…”

“…Previously, hexC had been localized to a 0.6-kb BclI-NcoI chromosomal fragment (Fig. 2) which failed to complement mutants deficient in GLK or EDD activity (57). To further delineate the hexC locus, plasmid pPZ196 (Fig.…”

“…Cloning and complementation analyses revealed, however, that the four genes were expressed from separate promoters (15,57), indicating that the genes were not contained in a single operon. Two regulatory effects further defined this group of genes.…”

“…Two regulatory effects further defined this group of genes. The first was the hexR mutation, thus far inseparable from edd, which caused loss of inducibility of glk, zwf, edd, and eda (15,47,57). The second was a locus designated hexC (15), also closely linked to * Corresponding…”

“…Two regulatory effects further defined this group of genes. The first was the hexR mutation, thus far inseparable from edd, which caused loss of inducibility of glk, zwf, edd, and eda (15,47,57). The second was a locus designated hexC (15), also closely linked to DIVERGENT edd AND gap GENES OF P. AERUGINOSA pRO1836 (57) was constructed from pRO1817 by deletion of a Sall fragment from pRO1817 ( Fig.…”